The CRM197 protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from the diphtheria toxin. CRM197 is produced by C. diphtheriae infected by the nontoxigenic phage .beta.197.sup.tox- created by nitrosoguanidine mutagenesis of the toxigenic corynephage .beta. (Uchida, T. et al. 1971, Nature New Biology 233:8-11). The CRM197 protein has the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid for glycine) in the mature protein and eliminates the toxic properties of diphtheria toxin. The CRM197 protein is a safe and effective T-cell dependent carrier for saccharides and is currently being used in the Haemophilus influenzae type b oligosacharide CRM197 conjugate vaccine (HibTiter.TM.; Lederle Praxis Biologicals, Rochester, N.Y.).
Production of significant quantities of the CRM197 protein for use in vaccines has been hindered due to low protein abundance. Techniques have been developed to bolster the production of CRM proteins using double lysogens (Rappuoli, R., 1983, Applied Env. Microbio. 46:560-564; U.S. Pat. No. 4,925,792 issued to R. Rappuoli; and Rappuoli, R., 1983, J. Bacteriol. 153: 1202-1210) of the nontoxigenic corynephage .beta.197. Rappuoli reports yields of CRM197 from double lysogens up to three fold higher than the single lysogens. The production Levels of CRM197 by single lysogens are adequate but economically unsatisfactory for the production of vaccines which utilize CRM197 protein.
Introduction of multiple lysogens of the corynephage .beta. into Corynebacterium diphtheriae is a laborious screening process for identifying strains that can overproduce the CRM197 protein, diphtheria toxin or other CRM proteins that are cross-reactive with diphtheria toxin. In addition, this process is limited in its ability to manipulate protein expression using standard recombinant techniques. It would therefore be beneficial to develop a process that can generate significant quantities of diphtheria toxin and CRM proteins by increasing the gene copy number without the use of corynephage .beta.; or by increasing the production levels of these proteins from strains lysogenic for corynephage .beta..